A growing body of evidence, contributed to significantly by work from the previous funding cycle, has demonstrated that gap junction channels display a surprising level of selectivity for their permeants, dictated by the connexin composition of the channels. The basis of this selectivity is complex, but understanding it will be critical to defining the diverse role these intercellular channels play in normal and pathogenic cell function. It is likely that the principles governing selective permeabilities of these channels will be distinct from the much better studied ion channels, and will require a new generation of tools. The current proposal seeks to develop these tools and to apply them to a mapping of the molecular determinants of permeabilty through these channels. A comparative analysis of gap junction channels comprised of different connexins that display very different permeability properties should define the structural diversity within these pores that may underlie their different properties (Aim #1). Scanning and targetted mutagenic and labeling strategies will then be used in the different connexins to map selectivity determinants for size and charge of permeants, as well as mapping sites of affinity for natural permants (Aim #2). We also propose the first comprehensive comparison of peremability characteristics and pore structure of intercellular gap junction channels and their corresponding hemichannels on the cell surface (Aim #3). Initial evidence suggests that there may be significant differences between these two channel isoforms, and growing evidence for the physiological significance of hemichannels in several processes, including cell death via apoptosis, etc., makes understanding such differences critical. The comparative studies proposed here should also resolve a controversy in the literature over the actual nature of the pore lining. While different members of the gap junction family have been mapped as the genetic causes of a surprising variety of human diseases, from catarracts, skin disease and peripheral nerve paralysis to the most common form of hereditary sensorineural deafness, we still have almost no understanding of how damage to these genes can cause the phenotypes observed. The current work will make a significant stride in resolving this issue by defining the molecular structure of these intercellular pores and how this can regulate what goes through them.